Lipid metabolism and cell composition of the oleaginous yeast Apiotrichum curvatum grown at different carbon to nitrogen ratios

Apiotrichum curvatum ATCC 20509, an oleaginous yeast that can accumulate up to 60% of its cellular dry weight as intracellular lipid when grown with excess carbon, was grown in nitrogen-limited, balanced, and lactose-free medium with asparagine as nitrogen source and lactose as carbon source. Biomass and lipid accumulation were measured, cell composition was analyzed, and catalase activity was followed as marker enzyme for peroxisomes. The organism accumulated 54% of its dry weight as total cellular lipid when grown under nitrogen limitation and accumulated only 20-25% of its dry weight as lipid when grown in balanced medium. When starved for carbon, cells utilized endogenous lipid and carbohydrate as carbon and energy sources; the intracellular contents of lipid and carbohydrate decreased by 31 and 26%, respectively. Intracellular carbohydrates also seemed to be used as intermediates for lipid accumulation and lipid turnover. Catalase activity was strongly induced (over 10-fold increase in specific activity) when cells metabolized endogenous lipid. The lipid content of cells was inversely related to catalase activity and to intracellular protein or total nitrogen content. Lipid content showed no correlation with intracellular carbohydrate content.     

Influence of nutrients, hexadecane, and temporal variations on nitrification and exopolysaccharide composition of river biofilms

Biofilms were cultivated on polycarbonate strips in rotating annular reactors using South Saskatchewan River water during the fall of 1999 and the fall of 2001. The reactors were supplemented with carbon (glucose), nitrogen (NH(4)Cl), phosphorus (KH(2)PO(4)), or combined nutrients (CNP), with or without hexadecane. The impact of these treatments on nitrification and on the exopolysaccharide composition of river biofilms was determined. The results showed that the biofilms had higher NH4(+) oxidation, NO3(-) production, and N2O production activities in fall 1999 than fall 2001 when grown with CNP but had higher activities in fall 2001 than fall 1999 when grown with individual nutrients. The exopolysaccharide amounts and proportions were generally higher in fall 1999 than fall 2001, as a consequence of the higher nutrient levels in the river water in the first year of this study. The addition of P and especially CNP stimulated NH4(+) oxidation by the biofilms, showing a P limitation in this river ecosystem. The presence of hexadecane negatively affected these activities and lowered the amounts of exopolysaccharides in CNP and P biofilms in fall 1999 but increased the biofilm activities and exopolysaccharide amounts in CNP biofilm in fall 2001. Antagonistic, synergistic, and independent effects between nutrients and hexadecane were also observed. This study demonstrated that the biofilm autotrophic nitrification activity in the South Saskatchewan River was limited by P, that this activity and the exopolysaccharide amounts and proportions were dependent on the nutrient concentrations in the river water, and suggested that exopolysaccharides may play a protective role for biofilm microorganisms against toxic pollutants.     

Nutritional control of sexuality in Chlamydomonas reinhardi

1. Cells of Chlamydomonas reinhardi grown in the light or dark on standard medium require an additional exposure to light in the absence of a nitrogen source, in order to become sexually active. As the culture ages, the light requirement decreases. 2. This light requirement is a function of nitrogen depletion, as shown by the observation that cells from cultures grown to maturity on a low nitrogen medium in the light or in the dark, have no additional light requirement for zygote formation. The withholding of no other component of the medium has this effect. 3. In cells requiring light for zygote formation, the light can be supplied before the mating types are mixed, indicating that light is required, not for mating per se, but for the conversion of vegetative cells to gametes. 4. Gametes can be dedifferentiated to the vegetative state by any nitrogen compound which the cells can use for growth; and by further exposure to light in the absence of a nitrogen source, these vegetative cells can again become gametic. 5. Cells grown at different nitrogen levels become gametic at widely different cell concentrations of nitrogen and carbon and C/N ratios. 6. It is postulated that the role of light in gametic differentiation is indirect, providing by photosynthesis, energy for the mating process and carbohydrates to tie up excess nitrogenous reserves; and that the concentration of some particular nitrogen fraction or compound determines whether or not gametic differentiation is initiated.     

Variation in composition and yield of exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4

The exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4 under different growth conditions have been analyzed for sugar composition. The first use of ion chromatography for the quantitative determination of microbial exopolysaccharide composition is reported. Klebsiella sp. strain K32 produced a polymer composed of rhamnose, galactose, and mannose early in its fermentation. The composition of the polymer varied markedly depending on the growth stage of the organism. Klebsiella sp. strain K32 grown in a fermentor produced a polymer which was rich in mannose during early exponential growth in a complex medium, but in the late stationary phase it did not contain detectable levels of mannose. The rhamnose present in the polymer increased from 12 to 55% over the course of growth, whereas galactose decreased from 63 to 45%. A. calcoaceticus BD4 produced a polymer containing rhamnose, glucose, mannose throughout its growth and stationary phase. Klebsiella sp. strain K32 and A. calcoaceticus BD4 were grown on various carbon sources in shake flasks. The polymer yield and composition from both organisms were found to vary with the carbon source. The exopolysaccharide with the highest mannose composition was obtained by using rhamnose as a carbon source for both organisms. These and other data suggest that regulatory changes caused by growth on different substrates result in either the production of a different distribution of polymers or a change in exopolysaccharide structure.     

Factors affecting accumulation and degradation of curdlan, trehalose and glycogen in cultures of Cellulomonas flavigena strain KU (ATCC 53703)

Cellulomonas flavigena strain KU (ATCC 53703) is a cellulolytic, Gram-positive bacterium which produces large quantities of an insoluble exopolysaccharide (EPS) when grown in minimal media with a high carbon-to-nitrogen (C/N) ratio. Earlier studies proved the EPS is structurally identical to the linear β-1,3-glucan known as curdlan and provided evidence that the EPS functions as a carbon and energy reserve compound. We now report that C. flavigena KU also accumulates two intracellular, glucose-storage carbohydrates under conditions of carbon and energy excess. These carbohydrates were partially purified and identified as the disaccharide trehalose and a glycogen/amylopectin-type polysaccharide. A novel method is described for the sequential fractionation and quantitative determination of all three carbohydrates from culture samples. This fractionation protocol was used to examine the effects of C/N ratio and osmolarity on the accumulation of cellular carbohydrates in batch culture. Increasing the C/N of the growth medium caused a significant accumulation of curdlan and glycogen but had a relatively minor effect on accumulation of trehalose. In contrast, trehalose levels increased in response to increasing osmolarity, while curdlan levels declined and glycogen levels were generally unaffected. During starvation for an exogenous source of carbon and energy, only curdlan and glycogen showed substantial degradation within the first 24 h. These results support the conclusion that extracellular curdlan and intracellular glycogen can both serve as short-term reserve compounds for C. flavigena KU and that trehalose appears to accumulate as a compatible solute in response to osmotic stress.     

Variation in composition and yield of exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4.

The exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4 under different growth conditions have been analyzed for sugar composition. The first use of ion chromatography for the quantitative determination of microbial exopolysaccharide composition is reported. Klebsiella sp. strain K32 produced a polymer composed of rhamnose, galactose, and mannose early in its fermentation. The composition of the polymer varied markedly depending on the growth stage of the organism. Klebsiella sp. strain K32 grown in a fermentor produced a polymer which was rich in mannose during early exponential growth in a complex medium, but in the late stationary phase it did not contain detectable levels of mannose. The rhamnose present in the polymer increased from 12 to 55% over the course of growth, whereas galactose decreased from 63 to 45%. A. calcoaceticus BD4 produced a polymer containing rhamnose, glucose, mannose throughout its growth and stationary phase. Klebsiella sp. strain K32 and A. calcoaceticus BD4 were grown on various carbon sources in shake flasks. The polymer yield and composition from both organisms were found to vary with the carbon source. The exopolysaccharide with the highest mannose composition was obtained by using rhamnose as a carbon source for both organisms. These and other data suggest that regulatory changes caused by growth on different substrates result in either the production of a different distribution of polymers or a change in exopolysaccharide structure.     

Influence of fructose and glucose on the production of exopolysaccharides and the activities of enzymes involved in the sugar metabolism and the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772

 The effect of fructose and glucose on the growth, production of exopolysaccharides and the activities of enzymes involved in the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus grown in continuous culture was investigated. When grown on fructose, the strain produced 25 mg l^-1 exopolysaccharide composed of glucose and galactose in the ratio 1:2.4. When the carbohydrate source was switched to a mixture of fructose and glucose, the exopolysaccharide production increased to 80 mg l^-1, while the sugar composition of the exopolysaccharide changed to glucose, galactose and rhamnose in a ratio of 1:7.0:0.8. A switch to glucose as the sole carbohydrate source had no further effect. Analysis of the enzymes involved in the synthesis of sugar nucleotides indicates that in cell-free extracts of glucose-grown cells the activity of UDP-glucose pyrophosphorylase was higher than that in cell-free extracts of fructose-grown cells. The activities of dTDP-glucose pyrophosphorylase and the rhamnose synthetic enzyme system were very low in glucose-grown cultures but could not be detected in fructose-grown cultures. Cells grown on a mixture of fructose and glucose showed similar enzyme activities as cells grown on glucose. Analysis of the intracellular level of sugar nucleotides in glucose-grown cultures of L. delbrueckii subsp. bulgaricus showed the presence of UDP-glucose and UDP-galactose in a ratio of 3.3:1, respectively, a similar ratio and slightly lower concentrations were found in fructose-grown cultures. The lower production of exopolysaccharides in cultures grown on fructose may be caused by the more complex pathway involved in the synthesis of sugar nucleotides. The absence of activities of enzymes leading to the synthesis of rhamnose nucleotides in fructose-grown cultures appeared to result in the absence of rhamnose monomer in the exopolysaccharides produced on fructose. Received: 1 February 1996/Received revision: 31 May 1996/Accepted: 2 June 1996     

Glutathione formation in Penicillium chrysogenum: stimulatory effect of ammonium

Penicillium chrysogenum produced glutathione after growth in a defined medium containing 10 mM-NH4Cl as the sole source of nitrogen. The use of higher ammonium concentrations (100 mM) resulted in stimulation of growth and glutathione formation. In addition, increases in the intracellular pools of glutamate, alanine and glutamine, proportional to the amount of ammonium present in the medium were observed. Resting cell systems, prepared from cells previously grown with ammonium, were able to produce glutathione when incubated with ammonium or the amino acids glutamate, alanine and glutamine. A mutant lacking NADP-dependent glutamate dehydrogenase activity (which has a leaky phenotype on ammonium as sole nitrogen source) required glutamate to synthesize glutathione. Resting cell systems of this mutant, prepared from cells previously grown with ammonium, did not produce glutathione even when incubated with glutamate or glutamine. On the other hand, resting cell systems of this mutant produced glutathione if prepared from cells previously grown with glutamate. The addition of glutamate to resting cell systems of the wild-type strain stimulated the synthesis of gamma-glutamylcysteine synthetase, the first enzyme of glutathione biosynthesis.     

Indication that the Nitrogen Source Influences Both Amount and Size of Exopolysaccharides Produced by Streptococcus thermophilus LY03 and Modelling of the Bacterial Growth and Exopolysaccharide Production in a Complex Medium

Streptococcus thermophilus LY03 is a yogurt strain producing the same exopolysaccharide material in both milk and MRS broth. Actually, two types of exopolysaccharides are produced simultaneously. The two exopolysaccharides are identical in monomer composition (galactose and glucose in a 4:1 ratio) but differ in molecular size. Gel permeation chromatography revealed a high-molecular-mass exopolysaccharide (1.8 × 10⁶) and a low-molecular-mass exopolysaccharide (4.1 × 10⁵). Both exopolysaccharides can be isolated from the fermentation broth separately. The proportion in which they are produced is strongly dependent on the carbon/nitrogen ratio of the fermentation broth. A shift from a high-molecular-mass exopolysaccharide to a low-molecular-mass exopolysaccharide was observed with increasing initial complex nitrogen concentrations. All necessary biokinetic parameters to study the kinetics of S. thermophilus LY03 fermentations were obtained from a mathematical model which describes both S. thermophilus LY03 growth and exopolysaccharide production and degradation. The model is valid with various initial complex nitrogen concentrations and can be applied to simulate exopolysaccharide production in a milk medium.     

Physiology of biosurfactant synthesis by Rhodococcus species H13-A

The physiology of biosurfactant synthesis by a soil isolate, identified as a Rhodococcus species, is described. The biosurfactant is a surface-active glycolipid produced during the stationary growth phase of Rhodococcus species H13-A on n-alkanes and fatty alcohols in response to limiting ammonium ion concentrations. Hexadecane-grown cells produced increasing amounts of extracellular glycolipid when the carbon to nitrogen ratio (C/N) was increased from 1.7 to 3.4. The increase in extracellular glycolipid in hexadecane-grown cells correlated with a decrease in the interfacial tension of the spent growth medium to values less than 5?mN/m. Significant levels of extracellular glycolipid were not detected in the spent growth medium of cells grown on triglycerides, fatty acids, ethanol, organic acids, or carbohydrates. Rhodococcus species H13-A contains the three indigenous plasmids pMVS100, pMVS200, and pMVS300, with neither pMVS200 nor pMVS300 being involved in glycolipid synthesis or hexadecane dissimilation. The role of pMVS100 remains undetermined. Key words: biosurfactants, glycolipids, trehalose lipids, Rhodococcus.     

Initiation of Yeast Sporulation by Partial Carbon, Nitrogen, or Phosphate Deprivation

In this paper we show that partial deprivation of a carbon source, a nitrogen source, or phosphate in the presence of all other nutrients needed for growth initiates meiosis and sporulation of Saccharomyces cerevisiae homothallic strain Y55. For carbon deprivation experiments, cells were grown in synthetic medium (pH 5.5) containing an excess of one carbon source and then transferred to the same medium containing different concentrations of the same carbon source. In the case of transfer to different acetate concentrations, the log optical density at 600 nm increased at the previous rate until the cells had used up all of the acetate, whereupon the cells entered a stationary phase and did not sporulate. The same was observed with ethanol. In contrast, at different concentrations of dihydroxy-acetone or pyruvate, cells grew at different rates and sporulated optimally at intermediate concentrations (50 to 75 mM). The response to galactose was similar but reflected the presence of a low-affinity galactose transport system and the induction of a high-affinity galactose transport system. Cells could also sporulate when a glucose medium ran out of glucose, apparently because they initiated sporulation during the subsequent lag period and then used the produced ethanol as a carbon source. For phosphate deprivation experiments, cells growing with excess ethanol or pyruvate and phosphate were transferred to the same medium containing limiting amounts of phosphate. First, they used up the intracellular phosphate reserves for rapid growth, and then they sporulated optimally when an intermediate concentration (30 μM) of phosphate had been added to the medium. For nitrogen deprivation experiments, cells grown with excess acetate, ethanol, or pyruvate and NH₄⁺ were transferred to the same medium from which all nitrogen had been removed. These cells sporulated well in acetate medium but poorly in ethanol and pyruvate media. However, the sporulation frequency in the latter media could be increased greatly by adding intermediate concentrations (1 mM) of the slowly metabolizable amino acids glycine, histidine, or phenylalanine. If one assumes that the sporulation response to partial deprivation of carbon-, nitrogen-, or phosphorus-containing compounds reflects control by a single metabolite, the intracellular concentration of this metabolite may decide at the START position (G1 phase) of the cell cycle whether a/α cells enter mitosis or meiosis.     

Effects of Salt Stress on Carbohydrate Metabolism in Desert Soil Alga Microcoleus vaginatus Gom.

The effects of salt stress on carbohydrate metabolism in Microcoleus vaginatus Gom., a cyanobacterium isolated from desert algal crusts, were investigated in the present study. Extracellular total carbohydrates and exopolysaccharides （EPS） in the culture medium produced by M. vaginatus increased significantly during the growth phase and reached a maximum during the stationary phase. The production of extracellular carbohydrates also significantly increased under higher salt concentrations, which was attributed to an increase in low molecular weight carbohydrates. In the presence of NaCI, the production of cellular total carbohydrates decreased and photosynthetic activity was impaired, whereas cellular reducing sugars, water-soluble sugars and sucrose content and sucrose phosphate synthase activity increased, reaching a maximum in the presence of 200 mmol/L NaCI. These parameters were restored to original levels when the algae were transferred to a non-saline medium. Sodium and K＋ concentrations of stressed cells decreased significantly and H＋-ATPase activity increased after the addition of exogenous sucrose or EPS. The results suggest that EPS and sucrose are synthesized to maintain the cellular osmOtic equilibrium between the intra-and extracellular environment, thus protecting algal cells from osmotic damage, which was attributed to the selective exclusion of cellular Na＋ and K＋ by H＋-ATPase.     

Metabolic flux analysis ofBeijerinckia indica for PS-7 production

In order to investigate central metabolic changes inBeijerinckia indica, cells were grown on different carbon sources and intracellular flux distributions were studied under varying concentrations of nitrogen. Metabolic fluxes were estimated by combining material balances with extracellular substrate uptake rate, biomass formation rate, and exopolysaccharide (EPS) accumulation rate. Thirty-one metabolic reactions and 30 intracellular metabolites were considered for the flux analysis. The results revealed that most of the carbon source was directed into the Entner-Doudoroff pathway, followed by the recycling of triose-3-phosphate back to Hexose-6-phosphate. The pentose phosphate pathway was operated at a minimal level to supply the precursors for biomass formation. The different metabolic behaviors under varying nitrogen concentrations were observed with flux analysis.     

Effect of nitrogenous resource on growth,biochemical composition and ultrastructure of Isochrysis galbana (Isochrysidales,Haptophyta)

The nitrogenous resource used to promote algal growth has cost implications for mass culture processes. The present study therefore aimed to determine the effect of different nitrogenous resources (nitrate, ammonium and urea) on various performance parameters (growth, final cell yield, pigmentation, lipid yield and cellular and sub‐cellular characteristics) in Isochrysis galbana. Growth rate was unaffected by nitrogenous resource, but the final cellular yield in the nitrate and urea treatments far exceeded that evident in the ammonium treatments. The reduced cell yield in ammonium treatments and the earlier onset of the stationary phase was brought about by nitrogen‐starvation due to an increase in pH and resultant ammonia volatilization. This starvation initiated an early onset of lipid accumulation, chlorophyll depletion and an increase in the carotenoid to chlorophyll ratio relative to the other nitrogen (N) source treatments. Hence, in spite of being potentially the preferred source of N by algae (due to its reduced state), ammonium‐nitrogen is undesirable for mass culture. The performance parameters of Isochrysis grown in urea (an organic N source) and nitrate (an inorganic N source) were similar, but lipid accrued earlier in cells grown in medium supplemented with urea. This is advantageous for lipid acquisition for the production of biodiesel since it would reduce the duration of photobioreactor runs. Urea is easily available and considerably cheaper than all the other N sources tested and is thus recommended as the nitrogenous resource for large‐scale culture of I. galbana for biodiesel production.     

A prominent role for glucosylglycerol in the adaptation of Pseudomonas mendocina SKB70 to osmotic stress.

The mechanism of osmoadaptation in a salt-tolerant (1.2 M NaCl) bacterial isolate identified as Pseudomonas mendocina (N. J. Palleroni, M. Doudoroff, R. Y. Stanier, R. E. Solanes, and R. Mandel, J. Gen. Microbiol. 60:215-231, 1970) was investigated. In response to osmotic stress, this species accumulated a number of compatible solutes, the intracellular levels of which depended on both the osmolarity and the ionic composition of the growth medium. Glucosylglycerol [alpha-D-glucopyranosyl-alpha-(1-->2)-glycerol], N-acetylglutaminylglutamine amide, and L-alpha-glutamate were the major compatible solutes accumulated via de novo biosynthesis. Trehalose was also accumulated, but only in cells grown in the presence of high concentrations of sulfate or phosphate ions. Glycine betaine was accumulated only when supplied exogenously to cells grown at high osmolarity, and its accumulation caused a significant depletion of the intracellular pools of glucosylglycerol and glutamate. Glucosylglycerol was also found to accumulate in the type strains of P. mendocina and P. pseudoalcaligenes. This is the first report demonstrating the pivotal role of glucosylglycerol in osmoadaptation in a nonphotosynthetic microorganism.     

Effect of culture medium pH on bacterial gellan production.

The effect of the initial pH of the culture medium used in the production of the exopolysaccharide gellan by the bacterium Pseudomonas species ATCC 31461, when glucose or corn syrup served as the carbon source, was investigated. With glucose as the carbon source, exopolysaccharide formation was highest after 72 h of growth when the initial pH of the culture medium was 6.8 to 7.4. Polysaccharide production by the bacterial cells grown on corn syrup for 72 h was maximal when the initial pH of the medium was 7.0 or 7.2. Cell weights of the strain after 72 h tended to be higher for the glucose-grown cells than for the corn syrup-grown cells.     

Continuous Culture of Ruminal Microorganisms in Chemically Defined Medium: II. Culture Medium Studies

Ruminal ciliates have been grown in continuous culture in chemically defined media and in the absence of viable bacteria. Oligotrichic ruminal ciliates seem to require insoluble carbohydrates for growth; the holotrichic ciliates require soluble carbohydrates, but at low concentrations. Both groups of ciliates utilize amino acids as their principal nitrogen source when these are supplied in micromolar concentrations; at millimolar concentrations, amino acids are toxic, possibly from excessive ammonia formation arising from ciliate deaminase activity. Holotrichic ruminal ciliates are destroyed by overdeposition of amylopectin when glucose is present above 0.1% concentration in the medium. Ecological requirements of ruminal ciliates are also described.     

Exopolysaccharide production by the cyanobacterium Anabaena sp. ATCC 33047 in batch and continuous culture

The halotolerant, filamentous, heterocystous cyanobacterium Anabaena sp. ATCC 33047 released, during the stationary growth phase in batch culture and, at low dilution rate, in continuous culture, large amounts of an exopolysaccharide (EPS) to the culture medium. Different environmental, nutritional and physical parameters affected production and accumulation of the EPS. The presence of either a combined nitrogen source or NaCl at high concentration led to decreased EPS production, without affecting cell growth. In contrast, generation of the EPS was markedly enhanced in response to an increase in either air flow rate, temperature or irradiance. In continuous culture, accumulation of EPS in the medium increased in response to a decrease in the dilution rate, with maximal EPS productivity being reached at a dilution rate of 0.03 h⁻¹.     

Purification and characterization of adhesive exopolysaccharides from Pseudomonas putida and Pseudomonas fluorescens

In this study, the adhesive exopolysaccharides of strains of Pseudomonas putida and P. fluorescens, both isolated from freshwater epilithic communities, were examined with regard to their chemical composition, biosynthesis, and their role in adhesion. Electron microscopy showed that both strains were enrobed in fibrous glycocalyces and that these structures were involved in attachment of the cells to a solid surface and as structural matrices in the microcolony mode of growth. In batch culture experiments most of the extracellular polysaccharide of both strains was found to be soluble in the growth medium rather than being associated with bacterial cells. Exopolysaccharide was synthesized during all phases of growth, but when growth was limited by exhaustion of the carbon source, exopolysaccharide synthesis ceased whereas exopolysaccharide synthesis continued for some time after cessation of growth in nitrogen-limited cultures. Exopolysaccharide from both strains was isolated and purified. Pseudomonas putida synthesized an exopolysaccharide composed of glucose, galactose, and pyruvate in a ratio of 1:1:1; the P. fluorescens polymer contained glucose, galactose, and pyruvate in a ratio of 1:1:0.5, respectively. Polymers from both strains were acetylated to a variable degree.     

Glycine betaine and proline are the principal compatible solutes ofStaphylococcus aureus

The foodborne pathogenStaphylococcus aureus is distinguished by its ability to grow within environments of extremely high osmolarity (e.g., foods with low water activity values). In the present study, we examined the accumulation of intracellular organic solutes withinS. aureus strain ATCC 12600 when cells were grown in a complex medium containing high concentrations of NaCl. Consistent with previous reports [Measures JC (1975) Nature 257:398–400; Koujima I, et al. (1978) Appl Environ Microbiol 35:467–470; and Anderson CB, Witter LD (1982) Appl Environ Microbiol 43:1501–1503], intracellular proline was found to accumulate to high concentrations. However, NMR spectroscopy of cell extracts revealed glycine betaine to be the predominant intracellular organic solute accumulated within cells grown at high osmolarity. In additional experiments, we examined the growth rate ofS. aureus in a defined medium of high osmolarity and found it to be stimulated significantly by the presence of either exogenous proline or glycine betaine. Highest growth rates were obtained when the defined medium was supplemented with glycine betaine.